PROJECT SUMMARY Cancer cachexia is a debilitating syndrome that affects the vast majority of patients with advanced cancer and accounts for nearly 30% of cancer-related deaths. The lack of prognostic markers to identify patient susceptibility and effective treatment options represent major gaps in cancer cachexia knowledge. A key feature of cancer cachexia is the progressive depletion of skeletal mass, which is mediated in part by two secreted factors, Activin and Myostatin. Emerging studies have revealed that pharmacological inhibition of Activin/Myostatin signaling is sufficient to suppress cachexia and extend survival in several mouse models of cancer cachexia, raising the possibility that targeting this pathway might represent a promising strategy to curb cachexia and attendant morbidity and mortality in cancer patients. We have recently reported that overexpression of Twist1 in muscle progenitor cells causes severe muscle loss akin to cancer cachexia. Using several genetic mouse models of pancreatic cancer, we detected a massive increase in Twist1 expression in muscle undergoing cachexia. We also found that elevated levels of muscle Twist1 are associated with severe cachexia in cancer patients. Inactivation of Twist1, either genetically or pharmacologically, afforded substantial protection against cancer-mediated muscle cachexia, which translated into meaningful survival benefits. From a mechanistic perspective, we present evidence that tumor-derived Act-A induces expression of Twist1, which in turn drives expression of MuRF1 and Atrogin1, leading to muscle protein degradation and attendant cachexia. Finally, we found that Twist1 also induces Myostatin expression, further supporting its role in cancer-driven muscle cachexia. Based on our findings, we hypothesize that Twist1 might function in Activin/Myostatin signaling to coordinate a feed-forward loop to execute muscle cachexia during cancer progression. We also hypothesize that developing combinatorial therapeutic strategies targeting both Twist1 and Activin/Myostatin could mitigate potential drug toxicity by lowering the dose needed for each medicine and combat the development of resistance. These overarching hypotheses will be tested in the following three Specific Aims: Specific Aim 1: Investigate the role of Twist1 in cancer cachexia, focusing on its ability to mediate Activin-induced muscle depletion. Specific Aim 2: Explore the mechanisms by which Twist1 coordinates a feed-forward loop to sustain Activin/Myostatin-driven muscle loss during cancer cachexia progression. Specific Aim-3: Test the efficacy of combinatorial drugs regimens targeting both Twist1 and Activin/Myostatin signaling pathway in cancer cachexia. Comprehensive characterization of this newly discovered cachexia driver will likely open up new angles to the cachexia field, both in terms of understanding its mechanistic paradigms and in terms of drug discovery.